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The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies

THE ROLE OF ENDOPLASMIC RETICULUM CHAPERONES IN REGULATING
HEMATOPOIETIC STEM CELLS AND HEMATOLOGICAL MALIGNANCIES
by
Biquan Luo
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2013
Copyright 2013 Biquan Luo

The endoplasmic reticulum (ER) is a cellular organelle essential for protein folding and transportation, as well as lipid synthesis and Ca2⁺ storage. ER chaperones play critical roles in assisting protein folding and processing. Two most abundant ER chaperones are GRP94 and GRP78. ❧ Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. The adult BM niche maintains HSC quiescence via soluble cues and direct contact. Traditionally regarded as an ER chaperone that assists the folding and processing of secretory and membrane bound proteins, GRP94 is speculated to play an important part in regulating HSC homeostasis. To directly elucidate the requirement of GRP94 in HSC homeostasis, we employed an inducible conditional knockout strategy to eliminate GRP94 in the hematopoietic system. The mutant mice displayed a 2-fold increase in HSC pool size resulted from a loss of quiescence and an increase in proliferation. The expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the BM niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT controls, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Furthermore, GRP94-null HSCs exhibited a near complete loss of integrin α4 expression on the cell surface and showed impaired binding with fibronectin, which are key molecules known to mediate HSC-niche interactions, providing a possible explanation for the loss of interaction of GRP94-null HSCs with the adult BM niche. ❧ Further investigation into the underlying mechanisms for this intrinsic hyperproliferation in GRP94-defiecient BM HSCs discovered an 1.8-fold increase in AKT activation, corresponding with higher production of PI(3,4,5)P3, indicating elevated PI3K activation. Treatment of GRP94-null HSCs with AKT inhibitors demonstrated that this elevated AKT activation is required for the increased proliferation in GRP94-null HSCs. Microarray analysis on freshly isolated Lin⁻ c-Kit⁺ Sca-1⁺ (LSK) cells demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells. Furthermore, we observed a reduced expression of cell surface connexin 32 plaques in GRP94-null LSK cells. However, suppression of connexin 32 hemichannel activity in WT LSK cells through mimetic peptides did not lead to increased LSK proliferation in vitro. In addition, two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. ❧ The requirement of GRP94 for maintaining HSC-niche interaction provides a therapeutic strategy of targeting GRP94 in the treatment of multiple myeloma (MM), the progress of which relies closely on the interaction of tumor cells with the BM microenvironment. We utilized a syngeneic MM mouse model to test whether GRP94 knockdown in tumor cells suppresses disease progression. Knockdown of GRP94 in mouse MM cells with lentivirus-mediated shRNA led to increased apoptosis in vitro, and GRP94 protein expression was gradually restored despite the presence of puromycin selection. Despite the unsuccessful knockdown of GRP94 in vitro, the syngeneic MM mouse model was established via tail vein tumor cell inoculation. MM mice displayed clusters of cells with lymphocyte-like morphology in the BM, as well as lesions of bone resorption. Further analysis at late stage MM mice demonstrated multiple tumor formation in the BM, accompanied by significant weight loss and hunched posture. ❧ In parallel with the study on GRP94 in HSC homeostasis, this dissertation also includes studies on the role of GRP78 in regulating HSC proliferation and apoptosis under both physiological condition and PTEN-null driven leukemia progression. Unlike GRP94 whose depletion causes HSC hyperproliferation, GRP78 depletion did not affect the cell cycle distribution of HSCs. However, in the context of PTEN-null driven leukemia, partial reduction of GRP78 suppressed the hyperproliferation induced by PTEN inactivation. Examining the requirement of GRP78 for HSC survival and apoptosis revealed an increased HSC apoptosis upon complete loss of GRP78, whereas heterozygous deletion of Grp78 did not lead to increased HSC apoptosis even in the context of cancer. ❧ In summary, studies in this dissertation revealed the comprehensive roles of ER chaperones, namely GRP94 and GRP78, in regulating HSC homeostasis and hematological malignancies.

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THE ROLE OF ENDOPLASMIC RETICULUM CHAPERONES IN REGULATING
HEMATOPOIETIC STEM CELLS AND HEMATOLOGICAL MALIGNANCIES
by
Biquan Luo
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2013
Copyright 2013 Biquan Luo